Crystal structure of a thermophilic cytochrome P450 from the archaeon Sulfolobus solfataricus

The structure of the first P450 identified in Archaea, CYP119 from Sulfolobus solfataricus, has been solved in two different crystal forms that differ by the ligand (imidazole or 4-phenylimidazole) coordinated to the heme iron. A comparison of the two structures reveals an unprecedented rearrangement of the active site to adapt to the different size and shape of ligands bound to the heme iron. These changes involve unraveling of the F helix C-terminal segment to extend a loop structure connecting the F and G helices, allowing the longer loop to dip down into the active site and interact with the smaller imidazole ligand. A comparison of CYP119 with P450cam and P450eryF indicates an extensive clustering of aromatic residues may provide the structural basis for the enhanced thermal stability of CYP119. An additional feature of the 4-phenylimidazole-bound structure is a zinc ion tetrahedrally bound by symmetry-related His and Glu residues.     

Preliminary characterization and crystal structure of a thermostable cytochrome P450 from Thermus thermophilus

The second structure of a thermophile cytochrome P450, CYP175A1 from the thermophilic bacterium Thermus thermophilus HB27, has been solved to 1.8-A resolution. The overall P450 structure remains conserved despite the low sequence identity between the various P450s. The CYP175A1 structure lacks the large aromatic network found in the only other thermostable P450, CYP119, thought to contribute to thermal stability. The primary difference between CYP175A1 and its mesophile counterparts is the investment of charged residues into salt-link networks at the expense of single charge-charge interactions. Additional factors involved in the thermal stability increase are a decrease in the overall size, especially shortening of loops and connecting regions, and a decrease in the number of labile residues such as Asn, Gln, and Cys.     

Thermophilic cytochrome P450 (CYP119) from Sulfolobus solfataricus: high resolution structure and functional properties

Crystal structures of a thermostable cytochrome P450 (CYP119) and a site-directed mutant, (Phe24Leu), from the acidothermophilic archaea Sulfolobus solfataricus were determined at 1.5-2.0 A resolution. We identify important crystallographic waters in the ferric heme pocket, observe protein conformational changes upon inhibitor binding, and detect a unique distribution of surface charge not found in other P450s. An analysis of factors contributing to thermostability of CYP119 of these high resolution structures shows an apparent increase in clustering of aromatic residues and optimum stacking. The contribution of aromatic stacking was investigated further with the mutant crystal structure and differential scanning calorimetry.     

Protein thermostability: structure-based difference of residual properties between thermophilic and mesophilic proteins

Structure-based differences of residual properties between 20 pairs of thermophilic and mesophilic proteins were statistically analyzed to elucidate the factors governing protein thermostability. This study analyzed the distributions of outer residues, inner residues, flexible residues, rigid residues, hydrogen bonds, salt bridges, cation–pi interactions, and disulfide bonds in each protein in terms of residual structural states, which were determined as five kinds of states under residual packing value. Their structural patterns found in thermophilic protein groups were compared with those of mesophilic protein groups for showing distinctive difference of residual properties. The results of statistical tests (t-test) revealed that flexible residues in fully-exposed state and boundary state, salt bridges in exposed state, and hydrogen bonds in well-buried state could be critical factors related with protein thermostability. Such structure-based differences of residual properties would help to develop a strategy for enhancing protein thermostability.     

Role of loop dynamics in thermal stability of mesophilic and thermophilic adenylosuccinate synthetase: a molecular dynamics and normal mode analysis study

Enzymes from thermophiles are poorly active at temperatures at which their mesophilic homologs exhibit high activity and attain corresponding active states at high temperatures. In this study, comparative molecular dynamics (MD) simulations, supplemented by normal mode analysis, have been performed on an enzyme Adenylosuccinate synthetase (AdSS) from E. coli (mesophilic) and P. horikoshii (thermophilic) systems to understand the effects of loop dynamics on thermal stability of AdSS. In mesophilic AdSS, both ligand binding and catalysis are facilitated through the coordinated movement of five loops on the protein. The simulation results suggest that thermophilic P. horikoshii preserves structure and catalytic function at high temperatures by using the movement of only a subset of loops (two out of five) for ligand binding and catalysis unlike its mesophilic counterpart in E. coli. The pre-arrangement of the catalytic residues in P. horikoshii is well-preserved and salt bridges remain stable at high temperature (363K). The simulations suggest a general mechanism (including pre-arrangement of catalytic residues, increased polar residue content, stable salt bridges, increased rigidity, and fewer loop movements) used by thermophilic enzymes to preserve structure and be catalytically active at elevated temperatures.     

Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins.

Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures.     

cytochrome p450 superfamily in Arabidopsis thaliana: isolation of cDNAs,Differential Expression,and RFLP mapping of multiple cytochromes P450

We have isolated multiple cDNAs encoding cytochromes P450 (P450s) from Arabidopsis thaliana employing a PCR strategy. Degenerate oligonucleotide primers were designed from amino acid sequences conserved between two plant P450s, CYP71A1 and CYP73A2, including the heme-binding site and the proline-rich motif found in the N-terminal region, and 11 putative P450 fragments were amplified from first-strand cDNA from 7-day-old Arabidopsis as a template. With these PCR fragments as hybridization probes, 13 full-length and 3 partial cDNAs encoding different P450s have been isolated from an Arabidopsis cDNA library. These P450s have been assigned to either one of the established subfamilies: CYP71B, CYP73A, and CYP83A; or novel subfamilies: CYP76C, CYP83B, and CYP91A. The primary protein structures predicted from the cDNA sequences revealed that the regions around both the heme-binding site and the proline-rich motif were highly conserved among all these P450s. The N-terminal structures of the predicted P450 proteins suggested that these Arabidopsis P450s were located at the endoplasmic reticulum membrane. The loci of four P450 genes were determined by RFLP mapping. One of the clones, CYP71B2, was located at a position very close to the ga4 and gai mutations. RNA blot analysis showed expression patterns unique to each of the P450s in terms of tissue specificity and responsiveness to wounding and light/dark cycle, implicating involvement of these P450s in diverse metabolic processes.     

Structure-based analysis of five novel disease-causing mutations in 21-hydroxylase-deficient patients

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90-95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients.     

Aromatic stacking as a determinant of the thermal stability of CYP119 from Sulfolobus solfataricus

Two notable features of the thermophilic CYP119, an Arg154-Glu212 salt bridge between the F-G loop and the I helix and an extended aromatic cluster, were studied to determine their contributions to the thermal stability of the enzyme. Site-specific mutants of the salt bridge (Arg154, Glu212) and aromatic cluster (Tyr2, Trp4, Trp231, Tyr250, Trp281) were expressed and purified. The substrate-binding and kinetic constants for lauric acid hydroxylation are little affected in most mutants, but the E212D mutant is inactive and the R154Q mutant has higher K(s),K(m), and k(cat) values. The salt bridge mutants, like wild-type CYP119, melt at 91+/-1 degrees C, whereas mutation of individual residues in the extended aromatic cluster lowers the T(m) by 10-15 degrees C even though no change is observed on mutation of an unrelated aromatic residue. The extended aromatic cluster, but not the Arg154-Glu212 salt bridge, contributes to the thermal stability of CYP119.     

Stability and rigidity/flexibility—Two sides of the same coin?

Protein molecules require both flexibility and rigidity for functioning. The fast and accurate prediction of protein rigidity/flexibility is one of the important problems in protein science. We have determined flexible regions for four homologous pairs from thermophilic and mesophilic organisms by two methods: the fast FoldUnfold which uses amino acid sequence and the time consuming MDFirst which uses three-dimensional structures. We demonstrate that both methods allow determining flexible regions in protein structure. For three of the four thermophile–mesophile pairs of proteins, FoldUnfold predicts practically the same flexible regions which have been found by the MD/First method. As expected, molecular dynamics simulations show that thermophilic proteins are more rigid in comparison to their mesophilic homologues. Analysis of rigid clusters and their decomposition provides new insights into protein stability. It has been found that the local networks of salt bridges and hydrogen bonds in thermophiles render their structure more stable with respect to fluctuations of individual contacts. Such network includes salt bridge triads Agr-Glu-Lys and Arg-Glu-Arg, or salt bridges (such as Arg-Glu) connected with hydrogen bonds. This ionic network connects alpha helices and rigidifies the structure. Mesophiles can be characterized by stand alone salt bridges and hydrogen bonds or small ionic clusters. Such difference in the network of salt bridges results in different flexibility of homologous proteins. Combining both approaches allows characterizing structural features in atomic detail that determine the rigidity/flexibility of a protein structure. This article is a part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

The role of salt bridges on the temperature adaptation of aqualysin I,a thermostable subtilisin-like proteinase

Differences in salt bridges are believed to be a structural hallmark of homologous enzymes from differently temperature-adapted organisms. Nevertheless, the role of salt bridges on structural stability is still controversial. While it is clear that most buried salt bridges can have a functional or structural role, the same cannot be firmly stated for ion pairs that are exposed on the protein surface. Salt bridges, found in X-ray structures, may not be stably formed in solution as a result of high flexibility or high desolvation penalty. More studies are thus needed to clarify the picture on salt bridges and temperature adaptation. We contribute here to this scenario by combining atomistic simulations and experimental mutagenesis of eight mutant variants of aqualysin I, a thermophilic subtilisin-like proteinase, in which the residues involved in salt bridges and not conserved in a psychrophilic homolog were systematically mutated. We evaluated the effects of those mutations on thermal stability and on the kinetic parameters.Overall, we show here that only few key charged residues involved in salt bridges really contribute to the enzyme thermal stability. This is especially true when they are organized in networks, as here attested by the D17N mutation, which has the most remarkable effect on stability. Other mutations had smaller effects on the properties of the enzyme indicating that most of the isolated salt bridges are not a distinctive trait related to the enhanced thermal stability of the thermophilic subtilase.     

Molecular modeling and identification of substrate binding site of orphan human cytochrome P450 4F22

Cytochrome P450s are superfamily of heme proteins which generally monooxygenate hydrophobic compounds. The human cytochrome P450 4F22 (CYP4F22) was categorized into "orphan" CYPs because of its unknown function. CYP4F22 is a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4F22 remain unclear. In this study, a three-dimensional model of human P450 4F22 was constructed by comparative modeling using Modeller 9v5. The resulting model was refined by energy minimization subjected to the quality assessment from both geometric and energetic aspects and was found to be of reasonable quality. Docking approach was employed to dock arachidonic acid into the active site of CYP4F22 in order to probe the ligand-binding modes. As a result, several key residues were identified to be responsible for the binding of arachidonic acid with CYP4F22. These findings provide useful information for understanding the biological roles of CYP4F22 and structure-based drug design.     

Homology model of 1alpha,25-dihydroxyvitamin D3 24-hydroxylase cytochrome P450 24A1 (CYP24A1): active site architecture and ligand binding

Homology models of cytochrome P450 24A1 (CYP24A1) were constructed using three human P450 structures, CYP2C8, CYP2C9 and CYP3A4 as templates for the model building. Using molecular operating environment (MOE) software the lowest energy CYP24A1 model was then assessed for stereochemical quality and side chain environment. Further active site optimisation of the CYP24A1 model built using the CYP3A4 template was performed by molecular dynamics to generate a final CYP24A1 model. The natural substrate, 1,25-dihydroxyvitamin D(3) (calcitriol) and the CYP24 inhibitor (R)-N-(2-(1H-imidazol-1-yl)-2-phenylethyl)-4'-chlorobiphenyl-4-carboxamide ((R)-VID-400) were docked into the model allowing further validation of the active site architecture. Using the docking studies structurally and functionally important residues were identified with subsequent characterisation of secondary structure.     

Investigation of ligand selectivity in CYP3A7 by molecular dynamics simulations

Cytochrome P450 (CYP) 3A7 plays a crucial role in the biotransformation of the metabolized endogenous and exogenous steroids. To compare the metabolic capabilities of CYP3A7–ligands complexes, three endogenous ligands were selected, namely dehydroepiandrosterone (DHEA), estrone, and estradiol. In this study, a three-dimensional model of CYP3A7 was constructed by homology modeling using the crystal structure of CYP3A4 as the template and refined by molecular dynamics simulation (MD). The docking method was adopted, combined with MD simulation and the molecular mechanics generalized born surface area method, to probe the ligand selectivity of CYP3A7. These results demonstrate that DHEA has the highest binding affinity, and the results of the binding free energy were in accordance with the experimental conclusion that estrone is better than estradiol. Moreover, several key residues responsible for substrate specificity were identified on the enzyme. Arg372 may be the most important residue due to the low interaction energies and the existence of hydrogen bond with DHEA throughout simulation. In addition, a cluster of Phe residues provides a hydrophobic environment to stabilize ligands. This study provides insights into the structural features of CYP3A7, which could contribute to further understanding of related protein structures and dynamics.     

Comparative modeling and molecular docking of orphan human CYP4V2 protein with fatty acid substrates: Insights into substrate specificity

Cytochromes P450 (CYPs) are a super family of heme-containing enzymes well-known for their monooxgenase reaction. There are 57 CYP isoenzymes found in human which exhibit specific physiological functions. Thirteen members of this super family are classified as "orphan" CYP because of their unknown enzymatic functions. CYP4V2 is found to be a potential drug target for Bietti crystalline corneoretinal dystrophy (BCD). However, three-dimensional structure, the active site topology and substrate binding modes of CYP4V2 remain unclear. In this study, the three-dimensional model of CYP4V2 was constructed using the homology modeling method. Four possible fatty acid substrates namely, caprylic, lauric, myrisitc and palmitic acids were optimized and evaluated for drug likeness using Lipinski's rule of five. Further, these substrates were docked into active sites of CYP4V2 and several key residues responsible for substrate binding were identified. These findings will be helpful for the structure-based drug design and detailed characterization of the biological roles of CYP4V2.     

Contribution of salt bridges near the surface of a protein to the conformational stability

Salt bridges play important roles in the conformational stability of proteins. However, the effect of a surface salt bridge on the stability remains controversial even today; some reports have shown little contribution of a surface salt bridge to stability, whereas others have shown a favorable contribution. In this study, to elucidate the net contribution of a surface salt bridge to the conformational stability of a protein, systematic mutant human lysozymes, containing one Glu to Gln (E7Q) and five Asp to Asn mutations (D18N, D49N, D67N, D102N, and D120N) at residues where a salt bridge is formed near the surface in the wild-type structure, were examined. The thermodynamic parameters for denaturation between pH 2.0 and 4.8 were determined by use of a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. The denaturation Gibbs energy (DeltaG) of all mutant proteins was lower than that of the wild-type protein at pH 4, whereas there was little difference between them near pH 2. This is caused by the fact that the Glu and Asp residues are ionized at pH 4 but protonated at pH 2, indicating a favorable contribution of salt bridges to the wild-type structure at pH 4. Each contribution was not equivalent, but we found that the contributions correlate with the solvent inaccessibility of the salt bridges; the salt bridge contribution was small when 100% accessible, while it was about 9 kJ/mol if 100% inaccessible. This conclusion indicates how to reconcile a number of conflicting reports about role of surface salt bridges in protein stability. Furthermore, the effect of salts on surface salt bridges was also examined. In the presence of 0.2 M KCl, the stability at pH 4 decreased, and the differences in stability between the wild-type and mutant proteins were smaller than those in the absence of salts, indicating the compensation to the contribution of salt bridges with salts. Salt bridges with more than 50% accessibility did not contribute to the stability in the presence of 0.2 M KCl.     

Characterization of the oxygenated intermediate of the thermophilic cytochrome P450 CYP119.

Using UV-Vis, resonance Raman, and EPR spectroscopy we have studied the properties of the oxygenated ferrous cytochrome P450 from Sulfolobus solfataricus, (CYP119). The recently determined crystal structure of CYP119 is compared with other available structures of P450s, and detailed structural and spectroscopic analyses are reported. With several structural similarities to CYP102, such as in-plane iron position and a shorter iron-proximal ligand bond, CYP119 shows low-spin conformation preference in the ferric form and partially in the ferrous form at low temperatures. These structural features can explain the fast autoxidation of the oxyferrous complex of CYP119. Finally, we report the first UV-Vis and EPR spectra of the cryoradiolytically reduced oxygenated intermediate of CYP119. The primary reduced intermediate, a hydroperoxo-ferric complex of CYP119, undergoes a 'peroxide shunt' pathway during gradual annealing at 170-195 K and returns to the low-spin ferric form.     

Electrostatic strengths of salt bridges in thermophilic and mesophilic glutamate dehydrogenase monomers

Here we seek to understand the higher frequency of occurrence of salt bridges in proteins from thermophiles as compared to their mesophile homologs. We focus on glutamate dehydrogenase, owing to the availability of high resolution thermophilic (from Pyrococcus furiosus) and mesophilic (from Clostridium symbiosum) protein structures, the large protein size and the large difference in melting temperatures. We investigate the location, statistics and electrostatic strengths of salt bridges and of their networks within corresponding monomers of the thermophilic and mesophilic enzymes. We find that many of the extra salt bridges which are present in the thermophilic glutamate dehydrogenase monomer but absent in the mesophilic enzyme, form around the active site of the protein. Furthermore, salt bridges in the thermostable glutamate dehydrogenase cluster within the hydrophobic folding units of the monomer, rather than between them. Computation of the electrostatic contribution of salt bridge energies by solving the Poisson equation in a continuum solvent medium, shows that the salt bridges in Pyrococcus furiosus glutamate dehydrogenase are highly stabilizing. In contrast, the salt bridges in the mesophilic Clostridium symbiosum glutamate dehydrogenase are only marginally stabilizing. This is largely the outcome of the difference in the protein environment around the salt bridges in the two proteins. The presence of a larger number of charges, and hence, of salt bridges contributes to an electrostatically more favorable protein energy term. Our results indicate that salt bridges and their networks may have an important role in resisting deformation/unfolding of the protein structure at high temperatures, particularly in critical regions such as around the active site.     

Modelling human cytochromes P450 involved in drug metabolism from the CYP2C5 crystallographic template

A historical background to homology modelling of human P450s involved in drug metabolism is outlined, showing that the progress in crystallographic studies of bacterial forms of enzyme and, latterly, determination of a mammalian P450 crystal structure, has enabled the production of increasingly satisfactory models of human P450 enzymes. The methodology for the generation of P450 models by homology with crystallographic template structures is summarized, and recent results of CYP2C5-constructed models of P450s are described. These indicate that selective substrates are able to fit within the putative active sites of each enzyme, where key contacts with complementary amino acid residues are largely consistent with the results of site-directed mutagenesis experiments and metabolic studies. Consequently, the CYP2C5 crystal structure can be regarded at the current paradigm for homology modelling of the drug metabolizing P450s, especially those from the CYP2 family.     

Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase.

Protein engineering is a promising tool to obtain stable proteins. Comparison between homologous thermophilic and mesophilic enzymes from a given structural family can reveal structural features responsible for the enhanced stability of thermophilic proteins. Structures from pig heart cytosolic and Thermus flavus malate dehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequence homology, were compared with the aim of increasing cMDH stability using features from the Thermus flavus enzyme. Three potential salt bridges from Tf MDH were selected on the basis of their location in the protein (surface R176-D200, inter-subunit E57-K168 and intrasubunit R149-E275) and implemented on cMDH using site-directed mutagenesis. Mutants containing E275 were not produced in any detectable amount, which shows that the energy penalty of introducing a charge imbalance in a region that was not exposed to solvent was too unfavourable to allow proper folding of the protein. The salt bridge R149-E275, if formed, would not enhance stability enough to overcome this effect. The remaining mutants were expressed and active and no differences from wild-type other than stability were found. Of the mutants assayed, Q57E/L168K led to a stability increase of 0.4 kcal/mol, as determined by either guanidinium chloride denaturalization or thermal inactivation experiments. This results in a 15 degrees C shift in the optimal temperature, thus confirming that the inter-subunit salt bridge initially present in the T.flavus enzyme was formed in the cMDH structure and that the extra energy obtained is transformed into an increase in protein stability. These results indicate that the use of structural features of thermophilic enzymes, revealed by a detailed comparison of three-dimensional structures, is a valid strategy to improve the stability of mesophilic malate dehydrogenases.